Fanconi anemia (FA) is a rare, recessive disorder, characterized by progressive bone marrow failure, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer. FA has twelve complementation groups (FA-A, B, C, D1, D2, E, F, G, I, J, L, and M), the corresponding genes for eleven of which have been identified and their products participate in a DNA damage response network involving BRCA1 and BRCA2. We have successfully isolated a FA core complex that contains all five expected FA proteins, as well as four new components, including FANCL ubiquitin ligase and FANCB. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the FA-DNA damage response pathway. We are systematically investigating the remaining uncharacterized components of the complex as these proteins may be products of genes that are mutated in currently "uncomplemented" FA patients. Perhaps one most important discovery of our investigations is the identification of FANCM as a novel gene product that mediates the interaction between DNA damage response and the FA nuclear core complex. The primary hypothesis of this proposal is that the newly identified FANCM is the signal transducer of the entire FA core complex in response to DNA damage or genotoxic stress. In the first aim of this proposal we will investigate this hypothesis by functional complementation of the FANCM deficient cell line (EUFA867) and determine additional binding factors to gain more mechanistic insights into how FANCM functions in the FA-DNA repair pathway. Functions of these new FANCM binding factors in the FA-DNA repair pathway will be investigated by various biochemical and genetic approaches. We also hypothesize that FANCM activity is essential for the function of the FA-DNA repair pathway. In the second aim of this proposal we will analyze the functional properties of FANCM and use functional complementation to relate structure to function. Lastly we hypothesize that FANCM activity is regulated in vivo by DNA damage-induced hyperphosphorylation. In the third aim of this proposal we will use in vitro biochemical approaches to investigate candidate kinases that may regulate FANCM phosphorylation and hence activity. In addition, we will use complementation analysis to determine the functional consequences of altered phosphorylation. This study should yield insight into how the FA pathway is regulated, and ultimately, how it functions in the cellular response to DNA damage. Understanding the molecular basis of Fanconi anemia will contribute the prevention and treatment of this disease and of childhood leukemia.